Project Summary The central goal of this project is to identify the cellular mechanisms that underlie the mechanical hypersensitivity and pain in Fabry Disease (FD), and thereby identify novel targets for improved pain treatment. FD is the most common lysosomal storage disease. It results from a deficiency of the lysosomal enzyme ?- galactosidase A (?-Gal A) that leads to the accumulation of glycosphingolipids within the lysosomes of cells including dorsal root ganglia (DRG) neurons. Small fiber neuropathy is a hallmark of FD. Neuropathic-like pain begins around age 5, worsens with age, and affects 60-80% of all (male and female) patients. The pain is described as episodic ?pain crises? triggered by fever, exercise, fatigue or stress, and chronic ?permanent pain.? We created a transgenic rat model of FD using CRISPR/Cas9 to delete the gene encoding ?-Gal A. Fabry rats closely recapitulate many phenotypes observed in patients, including elevated serum levels of ?- galactosyl glycosphingolipids, spontaneous and mechanically-evoked pain behavior, pronounced lipid inclusions and aberrant accumulation of ?-galactosyl glycosphingolipids in small-diameter DRG neurons and severe hearing loss. Sensory neurons somata from Fabry rats have sensitized rapidly adapting mechanical currents and sensitized responses to mustard oil, suggesting that Piezo2 and TRPA1 channel activities may be enhanced. Inhibition of TRPA1 alleviates the behavioral mechanical hypersensitivity in FD. Afferent fibers also show clear spontaneous activity, which may underlie the ongoing pain. The Scientific Premise of this proposal is that DRG sensory neurons in FD are dysfunctional and that the glycosphingolipids elevated in FD sensitize Piezo2 and TRPA1 channels in sensory neurons, resulting in the mechanical hypersensitivity and ongoing pain. This proposal will define the mechanisms that mediate Fabry disease pain in a top-down approach through Aims that 1) define the stimulus evoked, ongoing, and crisis-evoked pain behavior over time in male and female Fabry rats, 2) determine if the DRG is a key site for the generation and maintenance of mechanical and ongoing pain, and 3) interrogate whether Piezo2 and/or TRPA1 ion channels mediate the mechanical sensitization observed in the Fabry rat. In addition to identifying pain mechanisms that are FD- specific, these studies will have a broader impact by identifying mechanisms that could advance the understanding of mechanisms that underlie tactile allodynia and spontaneous pain in other types of neuropathic disorders and uncover novel roles for lipids in mechanotransduction mechanisms in somatosensory systems.